Proton Affinitive Derivatization for Highly Sensitive Determination of Testosterone and Dihydrotestosterone in Saliva Samples by LC-ESI-MS/MS.

In this study, proton affinitive derivatization using picolinic acid and its analogs (3- and 6-methylpicolinic acid and 5-butylpicolinic acid) with proton affinitive moieties was performed for the highly sensitive determination of testosterone (T) and 5α-dihydrotestosterone (DHT) in saliva by LC-ESI-MS/MS. T and DHT were converted to their corresponding picolinate esters and their chromatographic behavior was investigated with a reversed phase column. The picolinate ester of each steroid exhibited a clear single peak and elution occurred in the following order: picolinate, 3/6-methylpicolinate, and 5-butylpicolinate.

Recent development of chemical derivatization in LC-MS for biomedical approaches.

LC-MS/MS is currently the most powerful system in biomedical analysis. At the same time, chemical derivatization is a useful technique to enhance the detection sensitivity of nonionizable or poorly ionizable molecules in LC-MS/MS. Derivatization improves the ionization efficiency, the chromatographic separation and/or the chemical stability.

Simultaneous quantification of salivary 3-hydroxybutyrate, 3-hydroxyisobutyrate, 3-hydroxy-3-methylbutyrate, and 2-hydroxybutyrate as possible markers of amino acid and fatty acid catabolic pathways by LC-ESI-MS/MS.

We have developed a highly sensitive and specific method for quantification of salivary 3-hydroxybutyrate (3HB), 3-hydroxyisobutyrate (3HIB), 3-hydroxy-3-methylbutyrate (3HMB) and 2-hydroxybutyrate (2HB), which could be new non-invasive biomarkers for catabolic pathways of fatty acids/ketogenic amino acids, valine, leucine, and methionine/threonine/α-ketobutyrate, respectively. The four hydroxybutyrates (3HB, 3HIB, 3HMB, and 2HB) were extracted from 5 µl of saliva, converted to 2-pyridylmethyl (2PM) ester derivatives, and measured by liquid chromatography-tandem mass spectrometry in positive electrospray ionization mode. [(13)C4]3HB was used as an internal standard.

Measurement of peripheral plasma 18-oxocortisol can discriminate unilateral adenoma from bilateral diseases in patients with primary aldosteronism.

Adrenal venous sampling is currently the only reliable method to distinguish unilateral from bilateral diseases in primary aldosteronism. In this study, we attempted to determine whether peripheral plasma levels of 18-oxocortisol (18oxoF) and 18-hydroxycortisol could contribute to the clinical differentiation between aldosteronoma and bilateral hyperaldosteronism in 234 patients with primary aldosteronism, including computed tomography (CT)-detectable aldosteronoma (n=113) and bilateral hyperaldosteronism (n=121), all of whom underwent CT and adrenal venous sampling. All aldosteronomas were surgically resected and the accuracy of diagnosis was clinically and histopathologically confirmed.

An innovative LC-MS/MS-based method for determining CYP 17 and CYP 19 activity in the adipose tissue of pre- and postmenopausal and ovariectomized women using 13C-labeled steroid substrates.

Context: Does adipose tissue produce steroid hormones like an endocrine organ?

Object: To clarify whether adipose tissue produces sex steroid hormone like an endocrine organ, we estimated several key steroid hormone levels, as well as CYP17 and CYP19 activity, in ovariectomized, pre- and postmenopausal women by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Subjects And Methods: The subjects were 19 premenopausal (n = 12), postmenopausal (n = 4), and ovariectomized women (n = 3) aged 27-68 years. Serum, visceral adipose and sc adipose samples were taken from these subjects and stored at -70°C.

Benzophenones from an endophytic fungus, Graphiopsis chlorocephala, from Paeonia lactiflora cultivated in the presence of an NAD+-dependent HDAC inhibitor.

Graphiopsis chlorocephala was separated from the surface-sterilized healthy leaves of Paeonia lactiflora (Paeoniaceae) and cultivated with nicotinamide (an NAD(+)-dependent HDAC inhibitor). The culture conditions significantly enhanced secondary metabolite production in the fungus and led to the isolation of a structurally diverse set of new benzophenones, cephalanones A-F (1-6), and a known 2-(2,6-dihydroxy-4-methylbenzoyl)-6-hydroxybenzoic acid (7). The structures of 1-6 were determined from NMR data, single crystal X-ray diffraction, and chemical transformations.

Structures and biomimetic synthesis of novel α-pyrone polyketides of an endophytic Penicillium sp. in Catharanthus roseus.

Novel polyketides, citreoviripyrone A (1) and B (2), known citreomontanin (3), and (-)-citreoviridin (4) were isolated from the mycelium of the endophytic fungus. The endophytic fungus, which belongs to the genus Penicillium, was separated from surface-sterilized healthy leaves of Catharanthus roseus. The structures of 1 and 2 were determined on the basis of NMR data, and 1 was characterized as an α-pyrone polyketide featuring bicyclo[4.

Tenuipyrone, a novel skeletal polyketide from the entomopathogenic fungus, Isaria tenuipes, cultivated in the presence of epigenetic modifiers.

The concomitant addition of the histone deacetylase inhibitor and the DNA methyltransferase inhibitor to the culture medium of an entomopathogenic fungus, Isaria tenuipes, greatly enhanced its secondary metabolite production and led to the isolation of tenuipyrone (1), a novel polyketide with an unprecedented tetracyclic ring system bearing a spiroketal structural component, along with two known C(10)-polyketides, cephalosporolide B (2), which is a plausible biosynthetic precursor of 1, and cephalosporolide F (3).

18-oxocortisol measurement in adrenal vein sampling as a biomarker for subclassifying primary aldosteronism.

Context: 18-Oxocortisol (18-oxoF) is a derivative of cortisol (F) that is produced by aldosterone synthase (CYP11B2). The potential for this steroid as a biomarker for differentiating patients with aldosterone-producing adenoma (APA) from those with idiopathic hyperaldosteronism (IHA) has not been examined.

Objectives: We measured 18-oxoF, aldosterone, and F in plasma from adrenal vein sampling (AVS) of patients with primary aldosteronism.

Highly sensitive and specific analysis of sterol profiles in biological samples by HPLC-ESI-MS/MS.

High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is a powerful method for the microanalysis of compounds in biological samples. Compared with gas chromatography-mass spectrometry (GC-MS), this method is more broadly applicable to various compounds and usually does not require a derivatization step before analysis. However, when neutral sterols are analyzed, the sensitivities of usual HPLC-MS/MS method are not superior to those of GC-MS because the sterols are relatively resistant to ionization.

Assay of labile estrogen o-quinones, potent carcinogenic molecular species, by high performance liquid chromatography-electrospray ionization tandem mass spectrometry with phenazine derivatization.

A sensitive and selective assay method for labile estrogen o-quinones, estrone (E(1))-2,3-quinone (Q), E(1)-3,4-Q, estradiol (E(2))-2,3-Q and E(2)-3,4-Q, based on the use of phenazine (Phz) derivatization with o-phenylenediamine and high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was described. The Phz derivatives of four estrogen o-quinones were purified by solid phase extraction and analyzed by HPLC-ESI-MS/MS. The protonated molecule was observed as a base peak for all Phz derivatives in their ESI-mass spectra (positive mode).

Probing the binding pocket of the active site of aromatase with 2-phenylaliphatic androsta-1,4-diene-3,17-dione steroids.

A series of 2-phenylaliphatic-substituted androsta-1,4-diene-3,17-diones (6) as well as their androstenedione derivatives (5) were synthesized as aromatase inhibitors to gain insights of structure-activity relationships of varying the alkyl moiety (C(1) to C(4)) of the 2-phenylaliphatic substituents as well as introducing a methyl- or trifluoromethyl function to p-position of a phenethyl moiety to the inhibitory activity. The inhibitors examined showed a competitive type inhibition. The 2-phenpropylandrosta-1,4-diene 6c was the most powerful inhibitor (K(i): 16.

Fusaric acid as a novel proton-affinitive derivatizing reagent for highly sensitive quantification of hydroxysteroids by LC-ESI-MS/MS.

A highly sensitive derivatization method for liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry of dehydroepiandrosterone (DHEA), testosterone (T), pregnenolone (P5), and 17alpha-OH-pregnenolone (17-OHP5) was developed based on the use of fusaric acid as a reagent. DHEA, P5, and 17-OHP5 were rapidly and quantitatively converted to the 3-fusarate esters by treatment with fusaric acid and 2-methyl-6-nitrobenzoic anhydride. The positive ESI-mass spectra of the fusarate esters of each steroid were dominated by the appearance of [M + H](+) as base peaks.

Development of highly sensitive quantification method for testosterone and dihydrotestosterone in human serum and prostate tissue by liquid chromatography-electrospray ionization tandem mass spectrometry.

We developed highly sensitive detection of testosterone (T) and 5alpha-dihydrotestosterone (DHT) by liquid chromatography-electrospray ionization tandem mass spectrometry using high proton affinitive derivatization of 17beta-hydroxyl group of T and DHT with picolinic acid, mobile phase consisting of MeCN-MeOH-H(2)O-formic acid and conventional octadecylsilica (ODS) column. Purification of the derivatives was carried out using solid-phase extraction with ODS cartridge. By this method, T and DHT were determined simultaneously with limits of quantification (LOQs) of 1 pg/0.

6beta,19-Bridged androstenedione analogs as aromatase inhibitors.

Inhibition of aromatase is an efficient approach for the prevention and treatment of breast cancer. New 6beta,19-bridged steroid analogs of androstenedione, 6beta,19-epithio- and 6beta,19-methano compounds 11 and 17, were synthesized starting from 19-hydroxyandrostenedione (6) and 19-formylandrost-5-ene-3beta,17beta-yl diacetate (12), respectively, as aromatase inhibitors. All of the compounds including known steroids 6beta,19-epoxyandrostenedione (4) and 6beta,19-cycloandrostenedione (5) tested were weak to poor competitive inhibitors of aromatase and, among them, 6beta,19-epoxy steroid 4 provided only moderate inhibition (K(i): 2.

Highly sensitive quantification of serum malonate, a possible marker for de novo lipogenesis, by LC-ESI-MS/MS.

We describe a new sensitive and specific method for the quantification of serum malonate (malonic acid, MA), which could be a new biomarker for de novo lipogenesis (fatty acid synthesis). This method is based upon a stable isotope-dilution technique using LC-MS/MS. MA from 50 microl of serum was derivatized into di-(1-methyl-3-piperidinyl)malonate (DMP-MA) and quantified by LC-MS/MS using the positive electrospray ionization mode.

Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS.

We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 microl) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode.

Aromatization of androstenedione and 16alpha-hydroxyandrostenedione in human placental microsomes. Kinetic analysis of inhibition by the 19-oxygenated and 3-deoxy analogs.

Inhibition of aromatase activity in human placental microsomes with androstenedione (AD) (1a) and its 19-oxygenated derivatives 1b and 1c, their 16alpha-hydroxy compounds 2 and 3, and 3-deoxyandrost-4-ene compounds 5 and 6 was studied using [1beta-(3)H]AD as a substrate and compared to that with [1beta-(3)H]16alpha-hydroxyandrostenedione (16-OHAD). AD series of steroids, compounds 1, inhibited competitively [1beta-(3)H]AD aromatization whereas other 16alpha-hydroxy steroids 2, 3, 5, and 6 inhibited AD aromatization in a non-competitive manner. On the other hand, all of 16-OHAD series, compounds 2, blocked the [1beta-(3)H]16-OHAD aromatization in a competitive manner whereas the AD series steroids 1 as well as the 3-deoxy-16alpha-hydroxy-17-one steroids 5 and 3-deoxy-16alpha,17beta-diol steroids 6 inhibited 16-OHAD aromatization non-competitively.

Development of sensitive derivatization method for aldosterone in liquid chromatography-electrospray ionization tandem mass spectrometry of corticosteroids.

A highly sensitive quantification method of aldosterone by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was investigated in a positive mode using recently developed picolinyl derivatization. Aldosterone was smoothly and quantitatively converted to the ethyl ether-picolinyl derivative by treatment with HCl-ethanol followed by the esterification with picolinic acid in the presence of 2-methyl-6-nitrobenzoic anhydride and 4-dimethylaminopyridine. The positive ion-ESI mass spectrum of the ethyl ether-picolinyl derivative was characterized by an appearance of protonated molecule ([M+H](+)) as a base peak.

Preparation and structural elucidation of the picolinyl ester of aldosterone for liquid chromatography-electrospray ionization tandem mass spectrometry.

Treatment of aldosterone with 35% HCl in EtOH or in MeOH followed by the picolinyl derivatization gave the picolinyl derivative of aldosterone-ethyl ether, 8, or methyl ether, 9, as a single and well-shaped liquid chromatographic peak. Picolinyl derivatization of aldosterone produced 21-picolinyl derivative of 18,20-anhydro-hemiacetal derivatives, 6, with poor chromatographic peak with wide half-width. Further conversion of 6 to 8 required long reaction time (>4 h).

Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS.

We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 mul of dried serum were derivatized into picolinyl esters (3beta-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously.